*Protein ID (NP or XP #) or Wolbachia#: LmjF.24.0370
*Organism (including strain): Leishmania major strain Friedlin
Etiologic Risk Group (see link below): Risk group 2 (RG2) - parasitic agents
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Leishmania major is a species of protozoa that cause zoonotic cutaneous leishmaniasis by infecting the macrophages and dendritic cells of the immune system. It is mostly found in Northwestern India, Northern Africa, and Northwestern China. It is transmitted by female sand flies and reproduces through binary fission once engulfed by the host’s macrophages. Symptoms of the disease involve lesions at the bitten areas and lasts around 3-6 months. While there is no cure yet, treatments include Fluconazole which can speed up recovery.
Essentiality of this protein:
Aspartate aminotransferases are involved in the final step of methionine regeneration from methylthioadenosine. Methionine is required for the initiation of protein synthesis, the biosynthesis of cysteine, phospholipids, and polyamines. It is also used for the methylation of rRNA and xenobiotics. Rapidly growing cells (which include those of parasites) synthesize large amounts of polyamines before DNA replication and inhibiting the formation of methionine can inhibit this process. In addition, since methionine is always the first amino acid in protein synthesis, a lack of methionine can inhibit this process as well.
Gene/Ortholog: Tb927.10.3660 (OG4_10189); Phenotype: no significant loss or gain of fitness in bloodstream forms (3 days); Source study: alsford
Gene/Ortholog: Tb927.10.3660 (OG4_10189); Phenotype: significant gain of fitness in bloodstream forms (6 days); Source study: alsford
Gene/Ortholog: Tb927.10.3660 (OG4_10189); Phenotype: no significant loss or gain of fitness in procyclic forms; Source study: alsford
Gene/Ortholog: Tb927.10.3660 (OG4_10189); Phenotype: no significant loss or gain of fitness in differentiation of procyclic to bloodstream forms; Source study: alsford
Gene/Ortholog: Tb11.02.2740 (OG4_10189); Phenotype: significant loss of fitness in bloodstream forms (3 days); Source study: alsford
Gene/Ortholog: Tb11.02.2740 (OG4_10189); Phenotype: no significant loss or gain of fitness in bloodstream forms (6 days); Source study: alsford
Gene/Ortholog: Tb11.02.2740 (OG4_10189); Phenotype: significant loss of fitness in procyclic forms; Source study: alsford
Gene/Ortholog: Tb11.02.2740 (OG4_10189); Phenotype: no significant loss or gain of fitness in differentiation of procyclic to bloodstream forms; Source study: alsford
--- List cost and quantity of substrate reagents, supplier, and catalog #
2 pack - Reaction Cocktail containing Reconstitute AST 10 Reagent, Stock No. 58-10, in 10 ml of deionized water ($96.82, ThermoScientific, TR70121)
1 pack - Type I, ammonium sulfate suspension, 200-500 units/mg protein, ($61.40, Sigma Aldrich, G2751-1KU)
Structure Available (PDB or Homology model): PDB #: 4H51
Current Inhibitors: Canaline, Carboxymethoxylamine
Expression Information (has it been expressed in bacterial cells): It has been expressed in E. Coli cells before.
Purification Method: Affinity chromatography - A solution containing 0.02 M potassium phosphate buffer with 250-300 mg of the enzyme is added to the hydroxyapatite column and eluted with the potassium phosphate buffer.
Image of protein (PyMol with features delineated and shown separately):
Figure 2 - Putative Aspartate Aminotransferase from Leishmania major (Friedlin strand). The protein is shown as cartoon and are colored from the N-terminal to the C-terminal using a rainbow color gradient.
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MLRHVCSSAVVAYSAGLAPVSCRRDGSTSYFSAVPRAPPDAIMGIAADFAKDMCPSKVNL
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences): **
*Target (protein/gene name): Putative Aspartate Aminotransferase
*NCBI Gene # or RefSeq#: 5652203
*Protein ID (NP or XP #) or Wolbachia#: LmjF.24.0370
*Organism (including strain): Leishmania major strain Friedlin
Etiologic Risk Group (see link below): Risk group 2 (RG2) - parasitic agents
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Leishmania major is a species of protozoa that cause zoonotic cutaneous leishmaniasis by infecting the macrophages and dendritic cells of the immune system. It is mostly found in Northwestern India, Northern Africa, and Northwestern China. It is transmitted by female sand flies and reproduces through binary fission once engulfed by the host’s macrophages. Symptoms of the disease involve lesions at the bitten areas and lasts around 3-6 months. While there is no cure yet, treatments include Fluconazole which can speed up recovery.
Link to TDR Targets page (if present): http://tdrtargets.org/targets/view?gene_id=29531
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.): http://www.ncbi.nlm.nih.gov/gene/5652203
Essentiality of this protein:
Aspartate aminotransferases are involved in the final step of methionine regeneration from methylthioadenosine. Methionine is required for the initiation of protein synthesis, the biosynthesis of cysteine, phospholipids, and polyamines. It is also used for the methylation of rRNA and xenobiotics. Rapidly growing cells (which include those of parasites) synthesize large amounts of polyamines before DNA replication and inhibiting the formation of methionine can inhibit this process. In addition, since methionine is always the first amino acid in protein synthesis, a lack of methionine can inhibit this process as well.
LmjF24.0370 has essentiality data
Gene/Ortholog: eco898 (OG4_10189); Phenotype: non-essential; Source study: blattner
Gene/Ortholog: eco898 (OG4_10189); Phenotype: non-essential; Source study: gerdes
Gene/Ortholog: eco898 (OG4_10189); Phenotype: non-essential; Source study: keio
Gene/Ortholog: eco898 (OG4_10189); Phenotype: non-essential; Source study: shigen
Gene/Ortholog: eco3899 (OG4_10189); Phenotype: non-essential; Source study: blattner
Gene/Ortholog: eco3899 (OG4_10189); Phenotype: essential; Source study: gerdes
Gene/Ortholog: eco3899 (OG4_10189); Phenotype: non-essential; Source study: keio
Gene/Ortholog: eco3899 (OG4_10189); Phenotype: non-essential; Source study: shigen
Gene/Ortholog: cel14208 (OG4_10189); Phenotype: Growth Defect; Source study: neb
Gene/Ortholog: Tb927.10.3660 (OG4_10189); Phenotype: no significant loss or gain of fitness in bloodstream forms (3 days); Source study: alsford
Gene/Ortholog: Tb927.10.3660 (OG4_10189); Phenotype: significant gain of fitness in bloodstream forms (6 days); Source study: alsford
Gene/Ortholog: Tb927.10.3660 (OG4_10189); Phenotype: no significant loss or gain of fitness in procyclic forms; Source study: alsford
Gene/Ortholog: Tb927.10.3660 (OG4_10189); Phenotype: no significant loss or gain of fitness in differentiation of procyclic to bloodstream forms; Source study: alsford
Gene/Ortholog: Tb11.02.2740 (OG4_10189); Phenotype: significant loss of fitness in bloodstream forms (3 days); Source study: alsford
Gene/Ortholog: Tb11.02.2740 (OG4_10189); Phenotype: no significant loss or gain of fitness in bloodstream forms (6 days); Source study: alsford
Gene/Ortholog: Tb11.02.2740 (OG4_10189); Phenotype: significant loss of fitness in procyclic forms; Source study: alsford
Gene/Ortholog: Tb11.02.2740 (OG4_10189); Phenotype: no significant loss or gain of fitness in differentiation of procyclic to bloodstream forms; Source study: alsford
Complex of proteins?: No
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
Mus musculus: http://www.ncbi.nlm.nih.gov/pubmed/11101353
*EC#: 2.6.1.1
Link to BRENDA EC# page: http://www.brenda-enzymes.org/php/result_flat.php4?ecno=2.6.1.1
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): Assay type: Spectrophotometric
-- link to Sigma (or other company) page for assay (see Sigma links below): http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/glutoxaltrans.pdf
-- links to assay reagents (substrates) pages. http://www.ncbi.nlm.nih.gov/pubmed/7379304
--- List cost and quantity of substrate reagents, supplier, and catalog #
2 pack - Reaction Cocktail containing Reconstitute AST 10 Reagent, Stock No. 58-10, in 10 ml of deionized water ($96.82, ThermoScientific, TR70121)
1 pack - Type I, ammonium sulfate suspension, 200-500 units/mg protein, ($61.40, Sigma Aldrich, G2751-1KU)
Structure Available (PDB or Homology model): PDB #: 4H51
Current Inhibitors: Canaline, Carboxymethoxylamine
Expression Information (has it been expressed in bacterial cells): It has been expressed in E. Coli cells before.
Purification Method: Affinity chromatography - A solution containing 0.02 M potassium phosphate buffer with 250-300 mg of the enzyme is added to the hydroxyapatite column and eluted with the potassium phosphate buffer.
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MLRHVCSSAVVAYSAGLAPVSCRRDGSTSYFSAVPRAPPDAIMGIAADFAKDMCPSKVNL
CIGVYRDEQNKPFVLESVRKAMSHIVERDTQMDYAPIAGLPSFVNSVQRLCFGKPMLDVQ
GDRIASAQTLSGTGALHLGVQLLQRSSGGSGTATLHIPSPSYPNHLNILQHLNVEASYYP
YYNLNTHRLNIEAMLNYLRQLPAGSVVLLHACAHNPTGCDPTPEEWQQIVDVICRSDLIP
FVDMAYQGFATGDIERDAYVLRALNQQEVPTYLVAQSLAKSFGLYGHRTGAVHIRCTTQK
EKANVLSQLQSTIRATYSNPPIFGARIADEILRTPQLRELWKSELNQMSSRLQDVRHRLV
AQLRACGSTRDWEYLEKGVGMMSLTGLTEEQVMALQQKYSVYLTRNGRIAFSGLSSENVA
YVAQSIHDVSR
*length of your protein in Amino Acids: 431
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 47.653 kDa
Molar Extinction coefficient of your protein at 280 nm wavelength: 44810 M-1 cm-1
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
1 atgcttcgtc acgtgtgctc gtccgccgtc gtggcctaca gtgctggcct ggcgccggtc
61 agctgccgcc gtgacgggtc gacgagctac ttctcggccg tgccgcgcgc gcctccagat
121 gccatcatgg gcattgctgc ggactttgcc aaggacatgt gcccaagcaa ggtaaacctt
181 tgcatcggag tctaccgtga tgagcagaac aagccctttg tgctggagtc tgtgcgcaag
241 gccatgtcgc acattgtgga gcgcgacacg cagatggact atgcacccat tgcgggtctg
301 ccgtccttcg tgaatagcgt gcagcggcta tgctttggaa agccaatgct ggacgttcaa
361 ggggaccgca tcgcctcggc acaaactctt agtggcaccg gtgccctgca cctcggcgtg
421 cagctactgc agcgctcctc tggtggctcc ggcaccgcga cgctgcacat cccgagcccc
481 tcctacccga atcacctgaa tatcctgcag catctaaacg ttgaggctag ctattacccg
541 tactacaatc tgaacacgca tcgcttgaac atcgaggcga tgctcaacta tctgcgccag
601 ctgcccgccg gctccgtggt cctgctgcac gcctgtgcgc acaacccgac tgggtgcgac
661 cctactccag aggaatggca gcagattgta gacgtcatct gccgaagcga tctcattccg
721 ttcgttgaca tggcatacca gggttttgcg acgggtgaca tcgagcgtga cgcctacgtc
781 ctacgcgcgt tgaatcagca agaggtcccc acgtacttgg tggcccagtc gttggcgaag
841 agctttggtc tctacgggca tcgcactggc gccgtccaca tccgctgcac cacgcagaag
901 gagaaggcaa atgtcctgtc gcagctgcag tcgacgattc gcgccaccta cagcaaccct
961 cccatcttcg gtgctcgcat cgcggacgaa attttgcgaa ccccgcagct gcgcgagctg
1021 tggaagtcgg agctgaacca gatgtcgagc cgactgcagg atgttcggca ccgcctcgtg
1081 gcccagctgc gtgcttgtgg ctcgacgcgc gactgggagt acctcgagaa aggtgtgggt
1141 atgatgtcgc tgacggggct gacggaggag caggtgatgg cgctgcagca gaagtacagc
1201 gtctacctga cgcgcaacgg tcgcatcgca ttttctggac tgagctccga aaatgtggcc
1261 tacgtcgctc agtccatcca cgatgtgtcc cggtag
*GC% Content for gene: 60.57%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
ATGCTGCGCC ATGTATGCAG CAGTGCGGTT GTCGCGTATT CCGCAGGTCT GGCCCCGGTG AGTTGCCGTC GCGACGGTAG TACTTCATAC TTCAGCGCGG TTCCGCGGGC ACCGCCGGAT GCCATTATGG GCATTGCCGC CGATTTTGCG AAAGATATGT GCCCGAGTAA GGTCAATCTG TGCATTGGAG TTTACCGCGA CGAACAGAAT AAACCATTTG TGCTGGAAAG TGTCCGGAAA GCGATGTCCC ACATCGTGGA ACGCGATACA CAAATGGATT ATGCTCCTAT TGCGGGGCTG CCCAGTTTTG TTAATAGCGT TCAGCGTCTG TGTTTCGGAA AACCTATGTT AGACGTACAG GGGGATCGCA TCGCCAGTGC TCAGACTCTG AGTGGCACCG GAGCACTGCA CCTGGGCGTT CAGCTTCTCC AACGGAGCTC GGGGGGGTCC GGGACGGCGA CTCTCCATAT CCCATCGCCG AGCTACCCTA ACCACCTTAA TATTCTCCAA CACCTGAACG TAGAGGCGTC GTACTATCCA TACTATAACC TCAACACCCA CCGTCTGAAT ATTGAAGCCA TGCTGAACTA TCTGCGCCAA TTACCGGCCG GCAGTGTTGT GCTGTTGCAT GCGTGTGCGC ACAACCCTAC TGGGTGCGAT CCGACTCCGG AGGAGTGGCA ACAGATCGTC GATGTCATCT GCCGCTCAGA TTTGATCCCG TTCGTCGATA TGGCCTACCA AGGTTTCGCA ACGGGGGATA TCGAACGGGA TGCTTATGTG TTGCGCGCGT TAAACCAGCA GGAAGTACCC ACCTATCTGG TGGCTCAGTC CCTGGCCAAG AGCTTTGGCC TGTACGGGCA TCGGACTGGG GCGGTGCACA TTCGGTGCAC TACACAGAAA GAAAAAGCAA ACGTCCTGTC CCAACTTCAA AGCACCATTC GCGCCACCTA TTCCAATCCC CCGATCTTCG GAGCCCGTAT TGCGGACGAG ATTTTGCGTA CTCCACAGCT GCGTGAACTG TGGAAATCAG AACTGAATCA AATGAGTTCT CGCTTACAGG ACGTCCGCCA CCGTCTCGTT GCGCAGCTTC GTGCCTGTGG CTCTACCCGC GATTGGGAGT ACCTGGAAAA AGGCGTCGGC ATGATGTCCC TCACGGGTCT GACTGAAGAG CAAGTAATGG CGCTGCAGCA AAAATATTCC GTGTATTTGA CGCGTAATGG CCGTATCGCT TTTAGTGGCT TAAGTTCAGA AAATGTAGCA TATGTAGCGC AGTCTATCCA TGATGTTTCC CGCTAA
*GC% Content for gene (codon optimized): 53.94%
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
**